This invention relates to the use of a non-mammalian DNA virus to express an exogenous gene in a mammalian cell.
Current methods for expressing an exogenous gene in a mammalian cell include the use of mammalian viral vectors, such as those that are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, or adeno-associated viruses. Other methods of expressing an exogenous gene in a mammalian cell include direct injection of DNA, the use of ligand-DNA conjugates, the use of adenovirus-ligand-DNA conjugates, calcium phosphate precipitation, and methods that utilize a liposome- or polycation-DNA complex. In some cases, the liposome- or polycation-DNA complex is able to target the exogenous gene to a specific type of tissue, such as liver tissue.
Typically, viruses that are used to express desired genes are constructed by removing unwanted characteristics from a virus that is known to infect, and replicate in, a mammalian cell. For example, the genes encoding viral structural proteins and proteins involved in viral replication often are removed to create a defective virus, and a therapeutic gene is then added. This principle has been used to create gene therapy vectors from many types of animal viruses such as retroviruses, adenoviruses, and herpes viruses. This method has also been applied to Sindbis virus, an RNA virus that normally infects mosquitoes but which can replicate in humans, causing a rash and an arthritis syndrome.
Non-mammalian viruses have been used to express exogenous genes in non-mammalian cells. For example, viruses of the family Baculoviridae (commonly referred to as baculoviruses) have been used to express exogenous genes in insect cells. One of the most studied baculoviruses is Autographa genes in insect cells. One of the most studied baculoviruses is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Although some species of baculoviruses that infect crustacea have been described (Blissard, et al., 1990, Ann. Rev. Entomology 35:127), the normal host range of the baculovirus AcMNPV is limited to the order lepidoptera. Baculoviruses have been reported to enter mammalian cells (Volkman and Goldsmith, 1983, Appl. and Environ. Microbiol. 45:1085-1093; Carbonell and Miller, 1987, Appl. and Environ. Microbiol. 53:1412-1417; Brusca et al., 1986, Intervirology 26:207-222; and Tjia et al., 1983, Virology 125:107-117). Although an early report of baculovirus-mediated gene expression in mammalian cells appeared, the authors later attributed the apparent reporter gene activity to the reporter gene product being carried into the cell after a prolonged incubation of the cell with the virus (Carbonell et al., 1985, J. Virol. 56:153-160; and Carbonell and Miller, 1987, Appl. and Environ. Microbiol. 53:1412-1417). These authors reported that, when the exogenous gene gains access to the cell as part of the baculovirus genome, the exogenous gene is not expressed de novo. Subsequent studies have demonstrated baculovirus-mediated gene expression in mammalian cells (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. 93:2348-2352). In addition to the Baculoviridae, other families of viruses naturally multiply only in invertebrates; some of these viruses are listed in Table 1.
Gene therapy methods are currently being investigated for their usefulness in treating a variety of disorders. Most gene therapy methods involve supplying an exogenous gene to overcome a deficiency in the expression of a gene in the patient. Other gene therapy methods are designed to counteract the effects of a disease. Still other gene therapy methods involve supplying an antisense nucleic acid (e.g., RNA) to inhibit expression of a gene of the host cell (e.g., an oncogene) or expression of a gene from a pathogen (e.g., a virus).
Certain gene therapy methods are being examined for their ability to correct inborn errors of the urea cycle, for example (see, e.g., Wilson et al., 1992, J. Biol. Chem. 267: 11483-11489). The urea cycle is the predominant metabolic pathway by which nitrogen wastes are eliminated from the body. The steps of the urea cycle are primarily limited to the liver, with the first two steps occurring within hepatic mitochondria. In the first step, carbamoyl phosphate is synthesized in a reaction that is catalyzed by carbamoyl phosphate synthetase I (CPS-I). In the second step, citrulline in formed in a reaction catalyzed by ornithine transcarbamylase (OTC). Citrulline then is transported to the cytoplasm and condensed with aspartate into arginosuccinate by arginosuccinate synthetase (AS). In the next step, arginosuccinate lyase (ASL) cleaves arginosuccinate to produce arginine and fumarate. In the last step of the cycle, arginase converts arginine into ornithine and urea.
A deficiency in any of the five enzymes involved in the urea cycle has significant pathological effects, such as lethargy, poor feeding, mental retardation, coma, or death within the neonatal period (see, e.g., Emery et al., 1990, In: Principles and Practice of Medical Genetics, Churchill Livingstone, N.Y.). OTC deficiency usually manifests as a lethal hyperammonemic coma within the neonatal period. A deficiency in AS results in citrullinemia which is characterized by high levels of citrulline in the blood. The absence of ASL results in arginosuccinic aciduria (ASA), which results in a variety of conditions including severe neonatal hyperammonemia and mild mental retardation. An absence of arginase results in hyperarginemia which can manifest as progressive spasticity and mental retardation during early childhood. Other currently used therapies for hepatic disorders include dietary restrictions; liver transplantation; and administration of arginine freebase, sodium benzoate, and/or sodium phenylacetate.
It has been discovered that a non-mammalian DNA virus carrying an exogenous gene expression construct and having an xe2x80x9calteredxe2x80x9d coat protein can be used to increase the efficiency with which the non-mammalian DNA virus expresses an exogenous gene in the mammalian cell. For example, expression of vesicular stomatitis virus glycoprotein G (VSV-G) as an altered coat protein on the surface of a virus particle of a baculovirus enhances the ability of the baculovirus to express an exogenous gene (e.g., a therapeutic gene) in a mammalian cell. While not being obligated to know or disclose the manner in which the invention works, nor being bound by any theory, it is postulated that the enhancement of gene expression is due to an increased ability of a virus particle to gain entry into the cytosol of the mammalian cell.
Accordingly, in one aspect, the invention features a method of expressing an exogenous gene in a mammalian cell(s), involving (i) introducing into the cell a non-mammalian DNA virus having an altered coat protein, the genome of which virus carries the exogenous gene under the control of a promoter that induces expression of the exogenous gene in the cell, and (ii) maintaining the cell under conditions such that the exogenous gene is expressed.
In a second aspect, the invention features a method of treating a gene deficiency disorder in a mammal (e.g., a human or a mouse), involving introducing into a cell (in vivo or ex vivo) a therapeutically effective amount of a non-mammalian DNA virus having an altered coat protein, the genome of which virus carries an exogenous gene, and maintaining the cell under conditions such that the exogenous gene is expressed in the mammal.
The invention further features a method for treating a tumor in a mammal, involving introducing into a cancerous cell of the mammal (e.g., a cancerous hepatocyte) a non-mammalian DNA virus (e.g., a baculovirus) having an altered coat protein, the genome of which virus expresses a cancer-therapeutic gene (encoding, e.g., a tumor necrosis factor, thymidine kinase, diphtheria toxin chimera, or cytosine deaminase). The exogenous gene can be expressed in a variety of cells, e.g., hepatocytes; cells of the central nervous system, including neural cells such as neurons from brain, spinal cord, or peripheral nerve; adrenal medullary cells; glial cells; skin cells; spleen cells; muscle cells; kidney cells; and bladder cells. Thus, the invention can be used to treat various cancerous or non-cancerous tumors, including carcinomas (e.g., hepatocellular carcinoma), sarcomas, gliomas, and neuromas. Included within the invention are methods for treating lung, breast, and prostate cancers. Either in vivo or in vitro methods can be used to introduce the virus into the cell in this aspect of the invention. Preferably, the exogenous gene is operably linked to a promoter that is active in cancerous cells, but not in other cells, of the mammal. For example, the xcex1-fetoprotein promoter is active in cells of hepatocellular carcinomas and in fetal tissue but it is otherwise not active in mature tissues. Accordingly, the use of such a promoter is preferred for expressing a cancer-therapeutic gene for treating hepatocellular carcinomas.
The invention also features a method for treating a neurological disorder (e.g., Parkinson""s Disease, Alzheimer""s Disease, or disorders resulting from injuries to the central nervous system) in a mammal. The method involves (a) introducing into a cell a therapeutically effective amount of a non-mammalian DNA virus (e.g., a baculovirus) having an altered coat protein, the genome of which virus includes an exogenous gene encoding a therapeutic protein, and (b) maintaining the cell under conditions such that the exogenous gene is expressed in the mammal. Particularly useful exogenous genes include those that encode therapeutic proteins such as nerve growth factor, hypoxanthine guanine phosphoribosyl transferase (HGPRT), tyrosine hydroxylase, dopadecarboxylase, brain-derived neurotrophic factor, basic fibroblast growth factor, sonic hedgehog protein, glial derived neurotrophic factor (GDNF) and RETLI (also known as GDNFRxcex1, GFR-1, and TRN1). Both neuronal and non-neuronal cells (e.g., fibroblasts, myoblasts, and kidney cells) are useful in this aspect of the invention. Such cells can be autologous or heterologous to the treated mammal. Preferably, the cell is autologous to the mammal, as such cells obviate concerns about graft rejection. Preferably, the cell is a primary cell, such as a primary neuronal cell or a primary myoblast.
In each aspect of the invention, the non-mammalian DNA virus is preferably an xe2x80x9cinvertebrate virusxe2x80x9d (i.e., a virus that infects, and replicates in, an invertebrate). For example, the DNA viruses listed in Table 1 can be engineered to have an altered coat protein(s) and used in the invention. Preferably, the invertebrate DNA virus is a baculovirus, e.g., a nuclear polyhedrosis virus, such as an Autographa californica multiple nuclear polyhedrosis virus. If desired, the nuclear polyhedrosis virus may be engineered such that it lacks a functional polyhedrin gene. Either or both the occluded form and budded form of virus (e.g., baculovirus) can be used. Another preferred virus is Bombyx mori Nuclear Polyhedrosis Virus (BmNPV).
The genome of the non-mammalian DNA virus can be engineered to include one or more genetic elements. In general, these elements are selected based on their ability to facilitate expression of (i) an altered coat protein on the surface of a virus particle, and/or (ii) an exogenous gene in a mammalian cell.
Any transmembrane protein that binds to a target mammalian cell, or that mediates membrane fusion to allow escape from endosomes, can be used as the altered coat protein on the non-mammalian DNA virus. Preferably, the altered coat protein is the polypeptide (preferably a glycosylated version) of a glycoprotein that naturally mediates viral infection of a mammalian cell (e.g., a coat protein of a mammalian virus, such as a lentivirus, and influenza virus, a hepatitis virus, or a rhabdovirus). Other useful altered coat proteins include proteins that bind to a receptor on a mammalian cell and stimulate endocytosis. Examples of suitable altered coat proteins include, but are not limited to, the coat proteins listed in Table 2, which are derived from viruses such as HIV, influenza viruses, rhabdoviruses, and human respiratory viruses. An exemplary vesicular stomatitis virus glycoprotein G (VSV-G) is encoded by the plasmid BV-CZPG, the nucleotide sequence of which is shown in FIG. 23. If desired, more than one coat protein can be used as altered coat proteins. For example, a first altered coat protein may be a tranrsmembrane protein that binds to a mammalian cell, and a second coat protein may mediate membrane fusion and escape from endosomes.
In a preferred embodiment, the altered coat protein is produced as a fusion (i.e., chimeric) protein. A particularly useful fusion protein includes (i) a transmembrane polypeptide (e.g., antibodies such as IgM, IgG, and single chain antibodies) fused to (ii) a polypeptide that binds to a mammalian cell (e.g., VCAM, NCAM, integrins, and selectins) or to a growth factor. Included among the suitable transmembrane polypeptides are various coat proteins that naturally exist on the surface of a non-mammalian or mammalian virus particle (e.g., baculovirus gp64, influenza hemagglutinin protein, and Vesicular stomatitis virus glycoprotein G). All or a portion of the transmembrane polypeptide can be used, provided that the polypeptide spans the membrane of the virus particle, such that the polypeptide is anchored in the membrane. Non-viral transmembrane polypeptides also can be used. For example, a membrane-bound receptor can be fused to a polypeptide that binds a mammalian cell and used as the altered coat protein. Preferably, the fusion protein includes a viral coat protein (e.g., gp64) and a targeting molecule (e.g., VSV-G). Fusion polypeptides that include all or a cell-binding portion of a cell adhesion molecule also are included within the invention (e.g., a gp64-VCAM fusion protein).
Typically, the gene encoding the altered coat protein is operably linked to a promoter that is not active in the mammalian cell to be infected with the virus but is active in a non-mammalian cell used to propagate the virus (i.e., a xe2x80x9cnon-mammalian-activexe2x80x9d promoter). By contrast, a mammalian-active promoter is used to drive expression of the exogenous gene of interest (e.g., a therapeutic gene), as is discussed below. Generally, promoters derived from viruses that replicate in non-mammalian cells, but which do not replicate in mammalian cells, are useful as non-mammalian active promoters. For example, when using a baculovirus as the non-mammalian DNA virus, a baculovirus polyhedrin promoter can be used to drive expression of the altered coat protein, since baculoviruses do not replicate in mammalian cells. Other examples of suitable non-mammalian active promoters include p10 promoters, p35 promoters, etl promoters, and gp64 promoters, all of which are active in baculoviruses. When insect cells are used to prepare a virus stock, this non-mammalian-active promoter allows the altered coat protein to be expressed on the surface of the resulting virus particles. Upon infecting a mammalian cell with the non-mammalian DNA virus having an altered coat protein, the polyhedrin promoter is inactive. Examples of suitable non-mammalian-active promoters for driving expression of altered coat proteins include baculoviral polyhedrin promoters (e.g., from pAcAb4 from Pharmingen, Inc.), p10 promoters (e.g., from pAcAb4 from Pharmingen, Inc.), p39 promoters (see Xu et al., 1995, J. Virol. 69:2912-2917), gp64 promoters (including TATA-independent promoters; see Kogan et al., 1995, J. Virol. 69:1452-1461), baculoviral IE1 promoters (see Jarvis et al., 1996, Prot. Expr. Purif. 8:191-203), and Drosophila alcohol dehydrogenase promoters (see Heberlein et al., 1995, Cell 41:965-977).
If desired, the non-mammalian-active promoter that is operably linked to the gene encoding the altered coat protein can be an inducible promoter that is activated in the non-mammalian cell in which the virus is propagated. Examples of suitable inducible promoters include promoters based on progesterone receptor mutants (Wang et al., 1994, Proc. Natl. Acad. Sci. 91:8180-8184), tetracycline-inducible promoters (Gossen et al., 1995, Science 268:1766-1760; 1992, Proc. Natl. Acad. Sci. 89:5547-5551, available from Clontech, Inc.), rapamycin-inducible promoters (Rivera et al., 1996, Nat. Med. 2:1028-1032), and ecdysone-inducible promoters (No et al., 1996, Proc. Nati. Acad. Sci. 93:3346-3351).
In principle, an inducible promoter that can be activated in either a non-mammalian or mammalian cell can be used in this embodiment of the invention, although in practice an inducer of the promoter typically would be added to the non-mammalian cell in which the virus is propagated, rather than the mammalian cell in which the exogenous gene is expressed. As an example, a gene encoding an altered coat protein can be operably linked to a promoter that is inducible by ecdysone (No et al., 1996, Proc. Natl. Acad. Sci. 93:3346-3351). In this case, the genome of the non-mammalian DNA virus is engineered to include a paired ecdysone response element operably linked to the gene encoding the altered coat protein. Expression of a heterodimeric ecdysone receptor in the presence of ecdysone (or an ecdysone analog) that is added to the cell activates gene expression from a promoter that is operably linked to a gene encoding an altered coat protein. The use of an inducible promoter to drive expression of the gene encoding the altered coat protein offers the advantage of providing an additional mechanism for controlling expression of the altered coat protein.
The genome of the non-mammalian DNA virus can be engineered to include additional genetic elements, such as a mammalian-active promoter of a long-terminal repeat of a transposable element or a retrovirus (e.g., Rous Sarcoma Virus); an inverted terminal repeat of an adeno-associated virus and an adeno-associated rep gene; and/or a cell-immortalizing sequence, such as the SV40 T antigen or c-myc. If desired, the genome of the non-mammalian DNA virus can include an origin of replication that functions in a mammalian cell (e.g., an Epstein Barr Virus (EBV) origin of replication or a mammalian origin of replication). Examples of mammalian origins of replication include sequences near the dihydrofolate reductase gene (Burhans et al., 1990, Cell 62:955-965), the xcex2-globin gene (Kitsberg et al., 1993, Cell 366:588-590), the adenosine deaminase gene (Carroll et al., 1993, Mol. Cell. Biol. 13:2927-2981), and other human sequences (see Krysan et al., 1989, Mol. Cell. Biol. 9:1026-1033). If desired, the origin of replication can be used in conjunction with a factor that promotes replication of autonomous elements, such as the EBNA1 gene from EBV. The genome of the non-mammalian DNA virus used in the invention can include a polyadenylation signal and an RNA splicing signal that functions in mammalian cells (i.e., a xe2x80x9cmammalian RNA splicing signal), positioned for proper processing of the product of the exogenous gene. In addition, the virus may be engineered to encode a signal sequence for proper targeting of the gene product.
The exogenous gene that is to be expressed in a mammalian cell is operably linked to a xe2x80x9cmammalian-activexe2x80x9d promoter (i.e., a promoter that directs transcription in a mammalian cell). Where cell-type specific expression of the exogenous gene is desired, the exogenous gene in the genome of the virus can be operably linked to a mammalian-active, cell-type-specific promoter, such as a promoter that is specific for liver cells, brain cells (e.g., neuronal cells), glial cells, Schwann cells, lung cells, kidney cells, spleen cells, muscle cells, or skin cells. For example, a liver cell-specific promoter can include a promoter of a gene encoding albumin, xcex1-1-antitrypsin, pyruvate kinase, phosphoenol pyruvate carboxykinase, transferrin, transthyretin, xcex1-fetoprotein, xcex1-fibrinogen, or xcex2-fibrinogen. Alternatively, a hepatitis virus promoter (e.g., hepatitis A, B, C, or D viral promoter) can be used. If desired, a hepatitis B viral enhancer may be used in conjunction with a hepatitis B viral promoter. Preferably, an albumin promoter is used. An a-fetoprotein promoter is particularly useful for driving expression of an exogenous gene when the invention is used to express a gene for treating a hepatocellular carcinoma. Other preferred liver-specific promoters include promoters of the genes encoding the low density lipoprotein receptor, xcex12-macroglobulin, xcex11-antichymotrypsin, xcex12-HS glycoprotein, haptoglobin, ceruloplasmin, plasminogen, complement proteins (C1q, C1r, C2, C3, C4, C5, C6, C8, C9, complement Factor I and Factor H), C3 complement activator, xcex2-lipoprotein, and xcex11-acid glycoprotein. For expression of an exogenous gene specifically in neuronal cells, a neuron-specific enolase promoter can be used (see Forss-Petter et al., 1990, Neuron 5: 187-197). For expression of an exogenous gene in dopaminergic neurons, a tyrosine hydroxylase promoter can be used. For expression in pituitary cells, a pituitary-specific promoter such as POMC may be useful (Hammer et al., 1990, Mol. Endocrinol. 4:1689-97). Typically, the promoter that is operably linked to the exogenous gene is not identical to the promoter that is operably linked to the gene encoding an altered coat protein.
Promoters that are inducible by external stimuli also can be used for driving expression of the exogenous gene. Such promoters provide a convenient means for controlling expression of the exogenous gene in a cell of a cell culture or within a mammal. Preferred inducible promoters include enkephalin promoters (e.g., the human enkephalin promoter), metallothionein promoters, mouse mammary tumor virus promoters, promoters based on progesterone receptor mutants, tetracycline-inducible promoters, rapamycin-inducible promoters, and ecdysone-inducible promoters. Methods for inducing gene expression from each of these promoters are known in the art.
Essentially any mammalian cell can be used in the invention; preferably, the mammalian cell is a human cell. The cell can be a primary cell (e.g., a primary hepatocyte, primary neuronal cell, or primary myoblast) or it may be a cell of an established cell line. It is not necessary that the cell be capable of undergoing cell division; a terminally differentiated cell can be used in the invention. If desired, the virus can be introduced into a primary cell approximately 24 hours after plating of the primary cell to maximize the efficiency of infection. Preferably, the mammalian cell is a liver-derived cell, such as a HepG2 cell, a Hep3B cell, a Huh-7 cell, an FTO2B cell, a Hepa1-6 cell, or an SK-Hep-1 cell) or a Kupffer cell; a kidney cell, such as a cell of the kidney cell line 293, a PC12 cell (e.g., a differentiated PC12 cell induced by nerve growth factor), a COS cell (e.g., a COS7 cell), or a Vero cell (an African green monkey kidney cell); a neuronal cell, such as a fetal neuronal cell, cortical pyramidal cell, mitral cell, a granule cell, or a brain cell (e.g., a cell of the cerebral cortex; an astrocyte; a glial cell; a Schwann cell); a muscle cell, such as a myoblast or myotube (e.g., a C2C12 cell); an embryonic stem cell, a spleen cell (e.g., a macrophage or lymphocyte); an epithelial cell, such as a HeLa cell (a human cervical carcinoma epithelial line); a fibroblast, such as an NIH3T3 cell; an endothelial cell; a WISH cell; an A549 cell; or a bone marrow stem cell. Other preferred mammalian cells include CHO/dhfrxe2x88x92 cells, Ramos, Jurkat, HL60, and K-562 cells.
The virus can be introduced into a cell in vitro or in vivo. Where the virus is introduced into a cell in vitro, the infected cell can subsequently be introduced into a mammal, if desired. Accordingly, expression of the exogenous gene can be accomplished by maintaining the cell in vitro, in vivo, or in vitro and in vivo, sequentially. Similarly, where the invention is used to express an exogenous gene in more than one cell, a combination of in vitro and in vivo methods may be used to introduce the gene into more than one mammalian cell.
If desired, the virus can be introduced into the cell by administering the virus to a mammal that carries the cell. For example, the virus can be administered to a mammal by subcutaneous, intravascular, or intraperitoneal injection. If desired, a slow-release device, such as an implantable pump, may be used to facilitate delivery of the virus to cells of the mammal. A particular cell type within the mammal can be targeted by modulating the amount of the virus administered to the mammal and by controlling the method of delivery. For example, intravascular administration of the virus to the portal, splenic, or mesenteric veins or to the hepatic artery may be used to facilitate targeting the virus to liver cells. In another method, the virus may be administered to cells or an organ of a donor individual (human or non-human) prior to transplantation of the cells or organ to a recipient.
In a preferred method of administration, the virus is administered to a tissue or organ containing the targeted cells of the mammal. Such administration can be accomplished by injecting a solution containing the virus into a tissue, such as skin, brain (e.g., the cerebral cortex), kidney, bladder, liver, spleen, muscle, thyroid, thymus, lung, or colon tissue. Alternatively, or in addition, administration can be accomplished by perfusing an organ with a solution containing the virus, according to conventional perfusion protocols.
In another preferred method, the virus is administered intranasally, e.g., by applying a solution of the virus to the nasal mucosa of a mammal. This method of administration can be used to facilitate retrograde transportation of the virus into the brain. This method thus provides a means for delivering the virus to brain cells, (e.g., mitral and granule neuronal cells of the olfactory bulb) without subjecting the mammal to surgery.
In an alternative method for using the virus to express an exogenous gene in the brain, the virus is delivered to the brain by osmotic shock according to conventional methods for inducing osmotic shock.
Where the cell is maintained under in vitro conditions, conventional tissue culture conditions and methods may be used. In a preferred method, the cell is maintained on a substrate that contains collagen, such as Type I collagen or rat tail collagen, or a matrix containing laminin. As an alternative to, or in addition to, maintaining the cell under in vitro conditions, the cell can be maintained under in vivo conditions (e.g., in a human). Implantable versions of collagen substrates are also suitable for maintaining the virus-infected cells under in vivo conditions in practicing the invention (see, e.g., Hubbell et al., 1995, Bio/Technology 13:565-576 and Langer and Vacanti, 1993, Science 260: 920-925).
The invention can be used to express a variety of exogenous genes encoding gene products such as a polypeptides or proteins, antisense RNAs, and catalytic RNAs. If desired, the gene product (e.g., protein or RNA) can be purified from the mammalian cell. Thus, the invention can be used in the manufacture of a wide variety of proteins that are useful in the fields of biology and medicine.
Where the invention is used to express an antisense RNA, the preferred antisense RNA is complementary to a nucleic acid (e.g., an mRNA) of a pathogen of the mammalian cell (e.g., a virus, a bacterium, or a fungus). For example, the invention can be used in a method of treating a hepatitis viral infection by expressing an antisense RNA that hybridizes to an mRNA of an essential hepatitis virus gene product (e.g., a polymerase mRNA). Other preferred antisense RNAs include those that are complementary to a naturally-occurring gene in the cell, which gene is expressed at an undesirably high level. For example, an antisense RNA can be designed to inhibit expression of an oncogene in a mammalian cell. Similarly, the virus can be used to express a catalytic RNA (i.e., a ribozyme) that inhibits expression of a target gene in the cell by hydrolyzing an mRNA encoding the targeted gene product. Antisense RNAs and catalytic RNAs can be designed by employing conventional criteria.
If desired, the invention can be used to express a dominant negative mutant in a mammalian cell. For example, viral assembly in a cell can be inhibited or prevented by expressing in that cell a dominant negative mutant of a viral capsid protein (see, e.g., Scaglioni et al., 1994, Virology 205:112-120; Scaglioni et al., 1996, Hepatology 24:1010-1017; and Scaglioni et al., 1997, J. Virol. 71:345-353).
The invention can be used to express any of various xe2x80x9ctherapeuticxe2x80x9d genes in a cell. A xe2x80x9ctherapeuticxe2x80x9d gene is one that, when expressed, confers a beneficial effect on the cell or tissue in which it is present, or on a mammal in which the gene is expressed. Examples of xe2x80x9cbeneficial effectsxe2x80x9d include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desirable characteristic. Included among the therapeutic genes are those genes that correct a gene deficiency disorder in a cell or mammal. For example, carbamoyl synthetase I can correct a gene deficiency disorder when it is expressed in a cell that previously failed to express, or expressed insufficient levels of, carbamoyl synthetase I. xe2x80x9cCorrectionxe2x80x9d of a gene deficiency disorder need not be equivalent to curing a patient suffering from a disorder. All that is required is conferral of a beneficial effect, including even temporary amelioration of signs or symptoms of the disorder. Also included are genes that are expressed in one cell, yet which confer a beneficial effect on a second cell. For example, a gene encoding insulin can be expressed in a pancreatic cell, from which the insulin is then secreted to exert an effect on other cells of the mammal. Other therapeutic genes include sequences that encode antisense RNAs nucleic acid that inhibit transcription or translation of a gene that is expressed at an undesirably high level. For example, an antisense gene that inhibits expression of a gene encoding an oncogenic protein is considered a therapeutic gene. xe2x80x9cCancer therapeuticxe2x80x9d genes are those genes that confer a beneficial effect on a cancerous cell or a mammal suffering from cancer. Particularly useful cancer therapeutic genes include the p53 gene, a herpes simplex virus thymidine kinase gene, and an antisense gene that is complementary to an oncogene.
The invention can be used to express a therapeutic gene in order to treat a gene deficiency disorder. Particularly appropriate genes for expression include those genes that are thought to be expressed at a less than normal level in the target cells of the subject mammal. Particularly useful gene products include carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, and arginase. Other desirable gene products include fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogen deaminase, factor VIII, factor IX, cystathione xcex2-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, xcex2-glucosidase, pyruvate carboxylase, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase (also referred to as P-protein), H-protein, T-protein, Menkes disease copper-transporting ATPase, Wilson""s disease copper-transporting ATPase, and CFTR (e.g., for treating cystic fibrosis).
The invention can also be used to express in a mammalian cell a gene that is expected to have a biological effect in mammals but not in insects (i.e., a xe2x80x9cmammal-specificxe2x80x9d gene). For example, a baculovirus genome can be used to express a mammalian myoD gene and thereby produce muscle proteins; such a gene would be expected to have a biological effect in mammalian cells but not insect cells. Other examples of mammal-specific genes include, but are not limited to, transcription factors that finction in mammalian, but not insect, cells. For example, the transcription factors c/ebp-alpha and chop10 will activate liver cell differentiation pathways when expressed from an insect genome (e.g., a baculovirus genome) in a mammalian cell. In contrast, expression of these mammal-specific transcription factors in an insect cell would be expected to have a minimal, or no, effect on the insect cell.
If desired, the vectors of the invention can be used to propagate genetic constructs in non-mammalian (e.g., insect) cells, with the advantage of inhibiting DNA methylation of the product. It has been observed that a promoter may become methylated in cell lines or tissues in which it is not normally expressed, and that such methylation is inhibitory to proper tissue specific expression (Okuse et al., 1997, Brain Res. Mol. Brain Res. 46:197-207; Kudo et al., 1995, J. Biol. Chem. 270:13298-13302). For example, a neural promoter may become methylated in a non-neural mammalian cell. By using, for example, insect cells (e.g., Sf9 cells) to propagate a baculovirus carrying an exogenous gene and a mammalian promoter (e.g., a neural promoter), the invention provides a means for inhibiting DNA methylation of the promoter prior to administration of the baculovirus and exogenous gene to the mammalian cell in which the exogenous gene will be expressed (e.g., a neural cell).
By xe2x80x9cnon-mammalianxe2x80x9d DNA virus is meant a virus that has a DNA genome (rather than RNA) and which is naturally incapable of replicating in a vertebrate, and specifically a mammalian, cell. Included are insect viruses (e.g., baculoviruses), amphibian viruses, plant viruses, and fungal viruses. Viruses that naturally replicate in prokaryotes are excluded from this definition. Examples of viruses that are useful in practicing the invention are listed in Table 1. As used herein, a xe2x80x9cgenomexe2x80x9d can include all or some of the nucleic acid sequences present in a naturally-occurring non-mammalian DNA virus. If desired, genes or sequences can be removed from the virus genome or disabled (e.g., by mutagenesis), provided that the virus retains, or is engineered to retain, its ability to express an exogenous gene in a mammalian cell. For example, the virus can be engineered such that it lacks a functional polyhedrin gene. Such a virus can be produced by deleting all or a portion of the polyhedrin gene from a virus genome (e.g., a baculovirus genome) or by introducing mutations (e.g., a frameshift mutation) into the polyhedrin gene so that the activity of the gene product is inhibited.
By xe2x80x9cinsectxe2x80x9d DNA virus is meant a virus that has a DNA genome and which is naturally capable of replicating in an insect cell (e.g., Baculoviridae, Iridoviridae, Poxviridae, Polydnaviridae, Densoviridae, Caulimoviridae, and Phycodnaviridae).
By xe2x80x9cexogenousxe2x80x9d gene or promoter is meant any gene or promoter that is not normally part of the non-mammalian DNA virus (e.g., baculovirus) genome. Such genes include those genes that normally are present in the mammalian cell to be infected; also included are genes that are not normally present in the mammalian cell to be infected (e.g., related and unrelated genes of other cells or species). As used herein, the term xe2x80x9cexogenous genexe2x80x9d excludes a gene encoding an xe2x80x9caltered coat protein.xe2x80x9d
By xe2x80x9caltered coat proteinxe2x80x9d is meant any polypeptide that (i) is engineered to be expressed on the surface of a virus particle, (ii) is not naturally present on the surface of the non-mammalian DNA virus used to infect a mammalian cell, and (iii) allows entry to a mammalian cell by binding to the cell and/or facilitating escape from the mammalian endosome into the cytosol of the cell. Typically, a gene encoding an altered coat protein is incorporated into the genome of the non-mammalian DNA virus used in the invention. If desired, a virus genome can be constructed such that the virus expresses a polypeptide that binds a mammalian receptor or counterreceptor on a mammalian cell. An altered coat protein can include all or a portion of a coat protein of a xe2x80x9cmammalianxe2x80x9d virus, i.e., a virus that naturally infects and replicates in a mammalian cell (e.g., an influenza virus). If desired, the altered coat protein can be a xe2x80x9cfusion protein,xe2x80x9d i.e., an engineered protein that includes part or all of two (or more) distinct proteins derived from one or multiple distinct sources (e.g., proteins of different species). Typically, a fusion protein used in the invention includes (i) a polypeptide that has a transmembrane region of a transmembrane protein (e.g., baculovirus gp64) fused to (ii) a polypeptide that binds a mammalian cell (e.g., an extracellular domain of VSV-G).
Although the term xe2x80x9calteredxe2x80x9d is used in reference to the coat protein (because it is altered in the sense that it is expressed on the surface of a virus particle on which it is not normally found), the protein itself need not differ in sequence or structure from a wild-type version of the protein. Thus, a wild-type tranrsmembrane protein that binds a mammalian cell can be used as the altered coat protein (e.g., a wild-type influenza virus hemagglutinin protein). Indeed, wild-type proteins are preferred. Nonetheless, non-wild-type proteins also can be used as the xe2x80x9calteredxe2x80x9d coat protein, provided that the non-wild-type coat protein retains the ability to bind to a mammalian cell. Examples of non-wild-type proteins include truncated proteins, mutant proteins (e.g., deletion mutants), and conservative variations of transmembrane polypeptides that bind a mammalian cell.
xe2x80x9cConservative variationxe2x80x9d denotes the replacement of an amino acid residue by another, functionally similar, residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as alanine, isoleucine, valine, leucine, or methionine, for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. The term xe2x80x9cconservative variationxe2x80x9d also includes the use of a substituted amino acid (i.e., a modified amino acid, such as Hydroxylysine) in place of an unsubstituted parent amino acid.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA sequence that includes the reference gene (e.g., the exogenous gene) is positioned adjacent to a DNA sequence that directs transcription of the DNA and, if desired, translation of the RNA (i.e., facilitates the production of the desired gene product).
By xe2x80x9cpromoterxe2x80x9d is meant at least a minimal sequence sufficient to direct transcription. A xe2x80x9cmammalian-activexe2x80x9d promoter is one that is capable of directing transcription in a mammalian cell. The term xe2x80x9cmammalian-activexe2x80x9d promoter includes promoters that are derived from the genome of a mammal, i.e., xe2x80x9cmammalian promoters,xe2x80x9d and promoters of viruses that are naturally capable of directing transcription in mammals (e.g., an MMTV promoter). Other promoters that are useful in the invention include those promoters that are sufficient to render promoter-dependent gene expression controllable for cell-type specificity, cell-stage specificity, or tissue-specificity (e.g., liver-specific promoters), and those promoters that are xe2x80x9cinduciblexe2x80x9d by external signals or agents (e.g., metallothionein, MMTV, and pENK promoters); such elements can be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene. The promoter sequence can be one that does not occur in nature, so long as it functions in a mammalian cell. An xe2x80x9cinduciblexe2x80x9d promoter is a promoter that, (a) in the absence of an inducer, does not direct expression, or directs low levels of expression, of a gene to which the inducible promoter is operably linked; or (b) exhibits a low level of expression in the presence of a regulating factor that, when removed, allows high-level expression from the promoter (e.g., the tet system). In the presence of an inducer, an inducible promoter directs transcription at an increased level.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) (e.g., a promoter) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9ccell-immortalizing sequencexe2x80x9d is meant a nucleic acid that, when present in a mammalian cell, is capable of transforming the cell for prolonged inhibition of senescence. Included are SV40 T-antigen, c-myc, telomerase, and E1A.
By xe2x80x9cantisensexe2x80x9d nucleic acid is meant a nucleic acid molecule (i.e., RNA) that is complementary (i.e., able to hybridize) to all or a portion of a target nucleic acid (e.g., a gene or mRNA) that encodes a polypeptide of interest. If desired, conventional methods can be used to produce an antisense nucleic acid that contains desirable modifications. For example, a phosphorothioate oligonucleotide can be used as the antisense nucleic acid in order to inhibit degradation of the antisense oligonucleotide by nucleases in vivo. Where the antisense nucleic acid is complementary to only a portion of the target nucleic acid encoding the polypeptide to be inhibited, the antisense nucleic acid should hybridize close enough to some critical portion of the target nucleic acid (e.g., in the translation control region of the non-coding sequence, or at the 5xe2x80x2 end of the coding sequence) such that it inhibits translation of a functional polypeptide (i.e., a polypeptide that carries out an activity that one wishes to inhibit (e.g., an enzymatic activity)). Typically, this means that the antisense nucleic acid should be complementary to a sequence that is within the 5xe2x80x2 half or third of a target mRNA to which the antisense nucleic acid hybridizes. As used herein, an xe2x80x9cantisense genexe2x80x9d is a nucleic acid that is transcribed into an antisense RNA. Typically, such an antisense gene includes all or a portion of the target nucleic acid, but the antisense gene is operably linked to a promoter such that the orientation of the antisense gene is opposite to the orientation of the sequence in the naturally-occurring gene.
The invention is useful for expressing an exogenous gene(s) in a mammalian cell in vitro or in vivo (e.g., a HepG2 cell). This method can be employed in the manufacture of proteins to be purified, such as proteins that are administered as pharmaceutical agents (e.g., insulin). The virus of the invention can also be used therapeutically. For example, the invention can be used to express in a patient a gene encoding a protein that corrects a deficiency in gene expression. In alternative methods of therapy, the invention can be used to express any protein, antisense RNA, or catalytic RNA in a cell.
The non-mammalian viral expression system of the invention offers several advantages. The altered coat protein on the virus enhances the ability of the non-mammalian DNA virus to infect and express a gene in a mammalian cell. Such a coat protein also can be used to confer cell-type specificity on the engineered virus. For example, expression of CD4+ on a cell enhances the ability of a virus expressing an HIV envelope gp120 protein to infect such CD4+ cells (Mebatsion et al., 1996, Proc. Natl. Acad. Sci. 93:11366-11370).
The invention allows for de novo expression of an exogenous gene; thus, detection of the exogenous protein (e.g., xcex2-galactosidase) in an infected cell represents protein that was actually synthesized in the infected cell, as opposed to protein that is carried along with the virus aberrantly. Because the non-mammalian viruses used in the invention are not normally pathogenic to humans and do not replicate in mammalian cells, concerns about safe handling of these viruses are minimized. Similarly, because the majority of naturally-occurring viral promoters are not normally active in a mammalian cell, production of undesired viral proteins is minimized. While traditional gene therapy vectors are based upon defective viruses that are propagated with helper virus or on a packaging line, the invention employs a virus that is not defective for growth on insect cells for purposes of virus propagation, but is intrinsically, and desirably, defective for growth on mammalian cells. Accordingly, in contrast to some mammalian virus-based gene therapy methods, the non-mammalian virus-based methods of the invention should not provoke a host immune response to proteins expressed by the virus in the mammalian cells.
The non-mammalian virus used in the invention can be propagated with cells grown in serum-free media, eliminating the risk of adventitious infectious agents occasionally present in the serum contaminating a virus preparation. In addition, the use of serum-free media eliminates a significant expense faced by users of mammalian viruses. Certain non-mammalian viruses, such as baculoviruses, can be grown to a high titer (i.e., 108 pfu/ml). Generally, the large virus genomes that can be used in the invention (e.g., the baculovirus genome at 130 kbp) can accept large exogenous DNA molecules (e.g., 100 kb). In certain embodiments, the invention employs a virus whose genome has been engineered to contain an exogenous origin of replication (e.g., the EBV oriP). The presence of such sequences on the virus genome allows episomal replication of the virus, increasing persistence in the cell. Where the invention is used in the manufacture of proteins to be purified from the cell, the invention offers the advantage that it employs a mammalian expression system. Accordingly, one can expect proper post-translational processing and modification (e.g., glycosylation) of the product of the exogenous gene.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.